Nano-particles for co-encapsulation of multiple enzymes and methods of preparation and use thereof

Abstract

The present application relates to the technical field of biological medicine, and in particular to a nano-particle for loading multi-enzyme combination, a preparation method and application thereof; the nano-particle for loading multi-enzyme combination comprises a core particle and a shell layer coated outside the core particle; wherein the core particle comprises a small molecule drug particle, a uricase and a catalase, the shell layer is a cross-linked layer and a target head for surface modification; the preparation method comprises mixing the drug nano-particle and a mixed enzyme solution with a saturated salt solution to obtain a metastable particle; and the metastable particle is solidified with a cross-linking agent. The nano-particle for loading multi-enzyme combination can improve the problem that H2O2, excessive accumulation of pathogens and inflammatory reaction cause damage to cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to nanoparticles encapsulating multiple enzymes, their preparation methods, and applications. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disorder caused by high levels of serum uric acid (SUA), which exists primarily as urate ions in the blood at a pH of approximately 7.4. High levels of uric acid can form MSU crystals that precipitate in the blood and deposit in joints or organs, thereby overstimulating the body's immune system and potentially triggering gout. Simultaneously, urate and urate crystals can deposit in the kidneys and other tissues, causing tissue damage. Furthermore, mild cases of hyperuricemia can cause renal arteriosclerosis and glomerular hypertension, disrupting the kidneys' autoregulation; severe cases may ultimately lead to glomerulosclerosis or uremia.

[0003] The drug treatments offered by related technologies include urate-lowering therapy (ULT), xanthine oxidase inhibitors (XO), and uricosurics, all of which work by increasing renal uric acid excretion. Among these, uricase, a high-molecular-weight drug, rapidly degrades uric acid and exhibits high activity. It can be coupled with polyethylene glycol (PEG) to form Pegloticase, a peptidase with low immunogenicity, high solubility, and a long serum half-life. However, uricase generates H2O2 during the oxidation of uric acid, and excessive accumulation of H2O2 can damage cells. Summary of the Invention

[0004] The purpose of this invention is to provide nanoparticles loaded with multiple enzymes, their preparation method and application, which can improve the problem of cell damage caused by excessive accumulation of H2O2 and pathogens and inflammatory response.

[0005] This invention is implemented as follows:

[0006] In a first aspect, the present invention provides a nanoparticle loaded with multiple enzymes, the nanoparticle being loaded with multiple enzymes comprising a core particle and a shell covering the core particle; wherein the core particle comprises a small molecule drug particle, uricase and catalase, and the shell is a cross-linked layer.

[0007] In an optional embodiment, the small molecule drug particles are curcumin protein particles; the cross-linking layer is a tannic acid / iron polymer layer.

[0008] In an optional embodiment, the nanoparticles encapsulating the multi-enzyme combination have a particle size of 230-250 nm.

[0009] In an optional embodiment, the mass ratio of enzyme to small molecule drug particles is 50:10-50:20.

[0010] In an optional embodiment, the crosslinking layer also has a modifying substance.

[0011] In a second aspect, the present invention provides a method for preparing nanoparticles loaded with multiple enzymes as described in any of the foregoing embodiments, comprising:

[0012] Drug nanoparticles and mixed enzyme solutions were mixed with saturated salt solutions to obtain metastable particles;

[0013] Metastable particles are cured with a crosslinking agent.

[0014] In an optional embodiment, the preparation method of drug nanoparticles includes: mixing a drug solution with a carrier and then sonicating.

[0015] In an optional embodiment, the carrier includes at least one of albumin, casein, Pluronic, PVA, and PVP.

[0016] In an optional embodiment, the crosslinking agent includes at least one of a crosslinking substance, dopamine, and acrylamide, wherein the crosslinking substance includes tannic acid and ferric chloride.

[0017] In an optional embodiment, the saturated salt solution includes at least one of ammonium sulfate solution, potassium sulfate solution, potassium phosphate solution, and sodium chloride solution.

[0018] In an optional embodiment, the method further includes: after curing the metastable particles with a crosslinking agent, incubating them with an incubator; wherein the target in the incubator includes one or more of peptides, proteins, antibodies, polysaccharides, and small molecule ligands.

[0019] Thirdly, the present invention provides the application of the multi-enzyme-coated nanoparticles as described in any of the foregoing embodiments, including: the application of the multi-enzyme-coated nanoparticles in medicaments for scavenging reactive oxygen species, protecting mitochondria, inhibiting inflammatory responses, degrading uric acid, preventing or treating hyperuricemia, preventing or treating oxidative stress damage, or inflammatory diseases.

[0020] The present invention has the following beneficial effects:

[0021] The method for preparing multi-enzyme-encapsulated nanoparticles of the present invention produces multi-enzyme-encapsulated nanoparticles that encapsulate the core particles in a shell, confining uricase and catalase within a limited space. This ensures that during the oxidation of uric acid to H2O2 by uricase, the H2O2 is efficiently and stably decomposed by catalase within the same confined space, thus mitigating the problem of excessive accumulation of uric acid and H2O2 that damages cells. Furthermore, the multi-enzyme-encapsulated nanoparticles of the present invention can be used in combination with small-molecule anti-inflammatory drugs to further improve inflammation-induced systemic damage. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a particle size characterization diagram of the nanoparticles in Experimental Example 1 of the present invention;

[0024] Figure 2 In Figure a, RAW246.7 cells exhibit different morphological characteristics in Experimental Example 2 of the present invention; in Figure b, cell safety test results are shown in Experimental Example 2 of the present invention; and in Figure c, NO release is shown at different drug ratios in Experimental Example 2 of the present invention.

[0025] Figure 3 This is a diagram showing the apoptosis of NRK-52e under different concentrations of UC and Cas-Cur intervention in the inflammatory culture medium of Experiment Example 3 of the present invention.

[0026] Figure 4 This is a graph showing the change in the apoptosis rate of NRK-52e by nanoparticles in the inflammatory culture medium in Experiment Example 4 of this invention;

[0027] Figure 5 This is a graph showing the intracellular ROS levels of NRK-52e cells after different drug treatments in the inflammatory culture medium in Experiment Example 5 of this invention.

[0028] Figure 6 This is a diagram showing the activity of mitochondria in NRK-52e cells after treatment with different drugs in the inflammatory culture medium in Experiment Example 6 of this invention.

[0029] Figure 7 This is a graph showing the release levels of TNF-α, IL-6, and IL-1β in the cell supernatant of Experiment Example 7 of this invention.

[0030] Figure 8This is a graph showing the serum UA levels of SD rats after injection of different drugs in Experiment Example 8 of this invention.

[0031] Figure 9 This is a stained section of rat kidney from Experiment Example 8 of the present invention, viewed under a 200× microscope.

[0032] Figure 10 The image of a stained section of rat kidney in Experiment Example 8 of this invention is shown under a 400× microscope. Figure 1 ;

[0033] Figure 11 The image of a stained section of rat kidney in Experiment Example 8 of this invention is shown under a 400× microscope. Figure 2 ;

[0034] Figure 12 The image of a stained section of rat kidney in Experiment Example 8 of this invention is shown under a 400× microscope. Figure 3 ;

[0035] Figure 13 The image of a stained section of rat kidney in Experiment Example 8 of this invention is shown under a 400× microscope. Figure 4 ;

[0036] Figure 14 The image of a stained section of rat kidney in Experiment Example 8 of this invention is shown under a 400× microscope. Figure 5 ;

[0037] Figure 15 The image of a stained section of rat kidney in Experiment Example 8 of this invention is shown under a 400× microscope. Figure 6 ;

[0038] Figure 16 The image of a stained section of rat kidney in Experiment Example 8 of this invention is shown under a 400× microscope. Figure 7 . Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0040] The present invention provides a nanoparticle loaded with multiple enzymes, comprising a core particle and a shell covering the core particle; wherein the core particle comprises small molecule drug particles, uricase and catalase, and the shell comprises a cross-linking layer and a modified target.

[0041] This multi-enzyme nanoparticle uses a shell to encapsulate the core particle, confining uricase and catalase within a limited space. During the process of uricase oxidizing uric acid to produce H2O2, H2O2 is decomposed by catalase, which is in the same limited space as uricase. This ensures the efficiency and stability of H2O2 decomposition and improves the problem of excessive H2O2 accumulation that can damage cells.

[0042] It should be noted that uricase can rapidly degrade uric acid, catalase can remove hydrogen peroxide produced by uricase and reactive oxygen species at the lesion site, and curcumin can inhibit the inflammatory response at the lesion site. The combination of these three can improve the therapeutic effect of the drug.

[0043] Optionally, the small molecule drug particles are curcumin protein particles. Curcumin is a natural hydrophobic polyphenol found in the rhizome of turmeric, which has various pharmacological activities such as anti-inflammatory and antioxidant effects. These activities are beneficial for treating kidney damage caused by hyperuricemia.

[0044] Of course, in other embodiments, the small molecule drug particles can also be polymer particles, such as polymer particles of curcumin.

[0045] Optionally, the cross-linking layer is a tannic acid / iron polymer layer; the tannic acid / iron polymer layer can serve as a drug delivery system to load the core particle drug, so that uricase, catalase, and curcumin are confined in a limited space, which not only effectively decomposes H2O2 and improves the problem of cell damage caused by excessive accumulation of H2O2, but also effectively utilizes curcumin for anti-inflammatory and antioxidant effects; moreover, the cross-linking of small molecule drug particles to form copolymers and solidify microparticles also gives the nanoparticles carrying multiple enzymes highly active reaction sites.

[0046] Optionally, the cross-linked layer may also have modifying substances, such as peptides, proteins, antibodies, polysaccharides, small molecule ligands, etc.; in this way, the duration of drug action can be enhanced, thereby improving drug efficacy.

[0047] Optionally, the nanoparticles used for encapsulating multiple enzymes are spherical, and the particle size of the nanoparticles used for encapsulating multiple enzymes is 230-250 nm, such as 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, etc.; nanoparticles with smaller particle sizes can better exert their corresponding effects.

[0048] Optionally, the mass ratio of enzyme to small molecule drug particles is 50:10-50:20, for example: 50:10, 50:13, 50:15, 50:17, 50:20, etc. Optimizing the mass ratio of enzyme to small molecule drug particles can effectively reduce uric acid and decompose H2O2 when using the nanoparticles loaded with multiple enzymes to improve hyperuricemia, and effectively improve the damage of inflammatory response to cells and kidneys.

[0049] This invention also provides a method for preparing nanoparticles loaded with multiple enzymes, comprising:

[0050] Drug nanoparticles and mixed enzyme solutions were mixed with saturated salt solutions to obtain metastable particles;

[0051] Metastable particles are cured with a crosslinking agent.

[0052] In this way, a shell can be formed to enclose the core particles, confining uricase and catalase within a limited space. During the process of uricase oxidizing uric acid to produce H2O2, H2O2 is decomposed by catalase, which is in the same limited space as uricase. This ensures the efficiency and stability of H2O2 decomposition and improves the problem of excessive accumulation of H2O2 that could damage cells.

[0053] Optionally, the preparation method of drug nanoparticles includes: mixing a drug solution with a carrier and then sonicating.

[0054] Optionally, the carrier includes at least one of albumin, casein, Pluronic, PVA, and PVP.

[0055] Optionally, the crosslinking agent includes at least one of the following: a crosslinking substance, dopamine, and acrylamide. The crosslinking substance includes tannic acid and ferric chloride, for example: tannic acid and ferric chloride, dopamine alone, acrylamide alone, tannic acid, ferric chloride, and dopamine used simultaneously, tannic acid, ferric chloride, and acrylamide used simultaneously, dopamine and acrylamide used simultaneously, etc., without specific limitations. When the crosslinking agent is dopamine or acrylamide, the crosslinked layer is not a tannic acid / ferric polymer layer.

[0056] Furthermore, during cross-linking and curing, tannic acid solution and ferric chloride solution can be added dropwise in sequence, or tannic acid solution and ferric chloride solution, as well as at least one of dopamine and acrylamide, can be added dropwise in sequence. Alternatively, all tannic acid solution can be added at once, followed by ferric chloride solution, or all tannic acid solution can be added at once, followed by ferric chloride solution, and then at least one of dopamine solution and acrylamide solution can be added. After adding the solution, the mixture can be shaken, and then centrifuged to remove unbound tannic acid, ferric chloride, etc.

[0057] Optionally, the saturated salt solution includes at least one of ammonium sulfate solution, potassium sulfate solution, potassium phosphate solution, and sodium chloride solution.

[0058] Optionally, the method for preparing the mixed enzyme solution includes: mixing an enzyme and a buffer solution, wherein the enzyme includes uricase and catalase; and the buffer solution includes one or more of phosphate buffer, carbonate buffer, and borate buffer.

[0059] The method for preparing multi-enzyme-coated nanoparticles of the present invention further includes: solidifying metastable particles with a crosslinking agent, and then incubating them with an incubator; wherein the target in the incubator includes one or more of peptides, proteins, antibodies, polysaccharides, and small molecule ligands to form long-cycle nanoparticles. In this way, protein modification on the surface of the crosslinked copolymer increases the cycling time of the nanoparticles.

[0060] Optionally, the target contains an active group, which is an amino group or a thiol group; further, the target is thiolized albumin.

[0061] The multi-enzyme-encapsulated nanoparticles of the present invention can be applied to drugs for scavenging reactive oxygen species, protecting mitochondria, inhibiting inflammatory responses, degrading uric acid, preventing or treating hyperuricemia, preventing or treating oxidative stress damage, or inflammatory diseases.

[0062] The present invention will be further described in detail below with reference to the embodiments.

[0063] Example 1

[0064] Weigh out 5 mg of curcumin (Cur) and 25 mg of sodium caseinate (Cas). Dissolve Cur in 100 μl of dimethyl sulfoxide (DMSO) to prepare a 50 mg / mL Cur stock solution. Dissolve Cas in 1 mL of pure water to prepare a 25 mg / mL casein solution. Add 40 μl of the Cur stock solution to 1 mL of the prepared Cas solution and vortex for 30 s to achieve a Cur concentration of 2 mg / mL. After vortexing, sonicate (150 W, 3 s sonication, 2 s interval, 5 min) to obtain curcumin nanoparticles (Cas-Cur).

[0065] Weigh 1 mg of uricase (URI) and 1 mg of catalase (CAT) and dissolve them in 1 ml of pH 8 borax-borate buffer to prepare a 1 mg / ml mixed enzyme solution. Salt out the mixture by mixing saturated ammonium sulfate solution, the mixed enzyme solution, and water at a volume ratio of 9:5:1 and vortex for 30 s to ensure homogeneity. After homogeneity, add 60 μl of curcumin nanoparticles (Cas-Cur) for salting out and vortex for 30 s. Add 20 μl of tannic acid (20 mg / ml) to the resulting solution and vortex for 30 s; then add 5 μl of ferric chloride solution (25 mg / ml), vortex for 30 s, centrifuge (14000 rpm, 10 min, 4 °C), discard the supernatant, resuspend in 1 ml of PBS buffer, centrifuge again, discard the supernatant, and sonicate on ice (150 W, 3 s sonication, 2 s interval, 8 min) to obtain the core particles UC / Cur.

[0066] Prepare 1 mL of 2.5 mg / mL bovine serum albumin (BSA) solution, add 15 μL of 10% Tris buffer and 25 μL of TCEP solution (32 mg / mL) to break the disulfide bonds of BSA to form thiol groups (-SH), and mix well to obtain BSA-SH. Resuspend the precipitate from the first centrifugation of UC / Cur in pure water, add 650 μL of BSA-SH solution, and incubate at 4 °C for 4 h. After incubation, centrifuge (14000 rpm, 10 min, 4 °C), discard the supernatant, add 1 mL of PBS buffer, and sonicate on ice (150 W, 3 s sonication, 2 s interval, 8 min) to obtain BSA-UC / Cur (nanoparticles for multi-enzyme coupling).

[0067] Example 2

[0068] Weigh out 5 mg of curcumin (Cur) and 25 mg of sodium caseinate (Cas). Dissolve Cur in 100 μl of dimethyl sulfoxide (DMSO) to prepare a 50 mg / mL Cur stock solution. Dissolve Cas in 1 mL of pure water to prepare a 25 mg / mL casein solution. Add 40 μl of the Cur stock solution to 1 mL of the prepared Cas solution and vortex for 30 s to achieve a Cur concentration of 2 mg / mL. After vortexing, sonicate (150 W, 3 s sonication, 2 s interval, 5 min) to obtain curcumin nanoparticles.

[0069] Weigh 1 mg of uricase (URI) and 1 mg of catalase (CAT) and dissolve them in 1 ml of pH 8 phosphate buffer to prepare a 1 mg / ml mixed enzyme solution. Salt out the mixture by mixing saturated potassium sulfate solution, the mixed enzyme solution, and water at a volume ratio of 9:5:1 and vortex for 30 s to ensure homogeneity. After homogeneity, add 60 μl of curcumin nanoparticles for salting out and vortex for 30 s. Add 20 μl of tannic acid (20 mg / ml) to the resulting solution and vortex for 30 s; then add 5 μl of ferric chloride solution (25 mg / ml) and 20 μl of dopamine solution (50 mg / ml), vortex for 30 s, centrifuge (14000 rpm, 10 min, 4 °C), discard the supernatant, resuspend in 1 ml of PBS buffer, centrifuge again, discard the supernatant, and sonicate on ice (150 W, 3 s sonication, 2 s interval, 8 min) to obtain the core particles.

[0070] Prepare 1 ml of 2.5 mg / mL bovine serum albumin (BSA) solution, add 15 μL of 10% Tris buffer and 25 μL of TCEP solution (32 mg / mL) to break the disulfide bonds of BSA to form thiol groups (-SH), and mix well to obtain BSA-SH. Resuspend the precipitate of the core particles after the first centrifugation in pure water, add 650 μL of BSA-SH solution, and incubate at 4 °C for 4 h. After incubation, centrifuge (14000 rpm, 10 min, 4 °C), discard the supernatant, add 1 ml of PBS buffer, and sonicate on ice (150 W, 3 s sonication, 2 s interval, 8 min) to obtain nanoparticles encapsulated with multiple enzymes.

[0071] Example 3

[0072] Weigh out 5 mg of curcumin (Cur) and 25 mg of sodium caseinate (Cas). Dissolve Cur in 100 μl of dimethyl sulfoxide (DMSO) to prepare a 50 mg / mL Cur stock solution. Dissolve Cas in 1 mL of pure water to prepare a 25 mg / mL casein solution. Add 40 μl of the Cur stock solution to 1 mL of the prepared Cas solution and vortex for 30 s to achieve a Cur concentration of 2 mg / mL. After vortexing, sonicate (150 W, 3 s sonication, 2 s interval, 5 min) to obtain curcumin nanoparticles.

[0073] Weigh 1 mg of uricase (URI) and 1 mg of catalase (CAT) and dissolve them in 1 ml of pH 8 borax-borate buffer to prepare a 1 mg / ml mixed enzyme solution. Salt out the mixture by mixing saturated potassium phosphate solution, the mixed enzyme solution, and water at a volume ratio of 9:5:1 and vortex for 30 s to ensure homogeneity. After homogeneity, add 60 μl of curcumin nanoparticles for salting out and vortex for 30 s. Add 20 μl of tannic acid (20 mg / ml) to the resulting solution and vortex for 30 s; then add 20 μl of acrylamide solution (50 mg / ml), vortex for 30 s, centrifuge (14000 rpm, 10 min, 4 °C), discard the supernatant, resuspend in 1 ml of PBS buffer, centrifuge again, discard the supernatant, and sonicate on ice (150 W, 3 s sonication, 2 s interval, 8 min) to obtain the core particles.

[0074] Prepare 1 ml of 2.5 mg / mL bovine serum albumin (BSA) solution, add 15 μL of 10% Tris buffer and 25 μL of TCEP solution (32 mg / mL) to break the disulfide bonds of BSA to form thiol groups (-SH), and mix well to obtain BSA-SH. Resuspend the precipitate of the core particles after the first centrifugation in pure water, add 650 μL of BSA-SH solution, and incubate at 4 °C for 4 h. After incubation, centrifuge (14000 rpm, 10 min, 4 °C), discard the supernatant, add 1 ml of PBS buffer, and sonicate on ice (150 W, 3 s sonication, 2 s interval, 8 min) to obtain nanoparticles encapsulated with multiple enzymes.

[0075] Experimental Example 1: Characterization of catalase-targeted nanoparticles.

[0076] The nanoparticles were characterized using a Malvern particle size analyzer and transmission electron microscopy. The particle size and Zeta film potential of the nanoparticles prepared in Example 1 are shown in Table 1. The polydispersity index (PDI) of both the nanoparticles and the polymer is <0.3, indicating a relatively uniform size distribution of the nanoparticles. Figure 1 As shown in the transmission electron microscopy (TEM) images, Cas-Cur is observed to be a uniform sphere, UC / Cur prepared by mixing with the enzyme is a particle composed of multiple proteins, and BSA-UC / Cur is coated with a layer of BSA on the outside. The above results suggest that enzyme drugs were successfully loaded into MOF and modified with BSA on the surface.

[0077] Table 1. Nanoparticle size and potential ( n=3)

[0078]

[0079] Experimental Example 2: Effects of different ratios of URI / CAT and Cas-Cur on NO production induced by uric acid / LPS in RAW246.7.

[0080] RAW246.7 cells in the logarithmic growth phase were harvested, and the cell density was adjusted to 7 × 10⁻⁶. 5 Cells were seeded at a rate of 1 ml of cell suspension per well in 12-well plates. The plates were incubated at 37°C with 5% CO2 for 24 h, and the cells were observed under a microscope to ensure they adhered to the well walls and covered the bottom. Culture media were prepared with 1 μg / ml LPS, 125 μg / ml MSU, and a mixture of MSU and LPS. Cells were divided into control, LPS, MSU, and LPS+MSU groups, with three replicates per group. Cells were treated with the corresponding drug-containing culture media according to their grouping and incubated for 24 h. Cell supernatant was collected, dead cells were removed by centrifugation, and NO content was determined using the Griess method.

[0081] The RAW246.7 cell inflammation model was observed under an optical microscope, such as... Figure 2 As shown in Figure a, RAW246.7 cells treated with LPS and uric acid (MSU) exhibited different states. Normal cells were densely distributed, rounded, and grew in an overlapping manner. With the addition of LPS and MSU, cell growth slowed significantly, and numerous sodium urate crystals coated the cells, resulting in irregular shapes and extended pseudopodia. After the addition of Cas-Cur, cell pseudopodia decreased significantly, but sodium urate crystals were not broken down. When the mixed enzyme was added, sodium urate crystals were broken down, no sodium urate crystals were observed in the field of view, and cell pseudopodia decreased significantly, indicating a marked improvement in cell state.

[0082] like Figure 2 As shown in Figure b, the nanoparticles exhibit low cytotoxicity to RAW246.7 cells and can be used for subsequent experiments. The Griess method was used to detect the effect of different ratios of Cur and mixed enzymes on NO secretion induced by MSU in RAW246.7 cells, and the optimal ratio was screened.

[0083] Statistical analysis was performed using SPSS 25.0, and the results are as follows: Figure 2 As shown in Figure c, compared with the model group, the combined use of UC and Cur significantly reduced NO levels than when used alone. When the ratio of enzyme to Cur was 25:10, the amount of NO released was significantly reduced (P < 0.05).

[0084] Experimental Example 3: The protective effect of different ratios of URI / CAT and Cas-Cur on apoptosis induced by inflammatory culture medium treatment in NRK-52e cells.

[0085] RAW246.7 cells were stimulated with LPS / MSU as described in Example 2 to create an inflammation culture medium. The supernatant was collected and fetal bovine serum was added to a concentration of 10% to prepare the inflammatory medium. Well-growing NRK-52e cells were digested and their density adjusted to 3 × 10⁻⁶ cells. 5 Cell suspension at 2 ml / well was seeded into 6-well plates. Cells were incubated at 37°C with 5% CO2 for 24 h, and cell adhesion was observed under a microscope. The old culture medium was discarded, and 2 ml of inflammatory medium was added for 1 h of stimulation. Cells were divided into control group, model group, and different ratios of URI / CAT and Cas-Cur, with 3 replicates per group. Different drugs were added to the cells according to the different groups, and the cells were incubated for 48 h. After culture, the supernatant was collected, and floating cells were collected by centrifugation. Cells were digested with trypsin and combined, then washed three times with PBS buffer before use. 1× Binding Buffer was prepared, and Annexin V-Cy5 and PI double staining working solution were prepared using 1× Binding Buffer. Finally, the cells were resuspended in 300 μL PBS and incubated at 4°C in the dark for 30 min before flow cytometry detection of cell apoptosis.

[0086] Flow cytometry was used to analyze apoptosis in cells under inflammatory culture conditions with different drug interventions to explore the ratio of enzyme to Cas-Cur. The protective effects of different ratios of UC groups (50 μg / ml or 25 μg / ml) and Cas-Cur (10 μg / ml or 5 μg / ml) on NRK-52e were investigated. Figure 3 As shown, the combination of the two drugs had a more significant protective effect on cells than single-drug administration. The cell survival rate at a ratio of 25:10 was significantly higher than that at 25:5 (P < 0.01), and the total mortality rate at 25:10 was also lower than that at 50:5 (P < 0.05). There was no significant difference in the total apoptosis rate of the enzyme between 50 μg / ml and 25 μg / ml (P > 0.05), and there was no significant difference in cell survival rate between 25:10 and 50:10 (P > 0.05). Considering the different NO secretion ratios, it is evident that a UC:Cas-Cur ratio of 25:10 is the most effective.

[0087] Experimental Example 4: The protective effect of nanoparticles against apoptosis induced by NRK-52e cells treated with inflammatory culture medium.

[0088] After digestion, the well-growing NRK-52e cells were counted and their density adjusted to 3 × 10⁻⁶. 5Cell suspension at 2 ml / well was seeded into 6-well plates. Cells were incubated at 37°C with 5% CO2 for 24 h, and cell adhesion was observed under a microscope. The old culture medium was discarded, and 2 ml of inflammatory medium was added for 1 h of stimulation. Cells were divided into control group, model group, Cas-Cur group, UC group, UC+Cas-Cur group, and BSA-UC / Cur group, with 3 replicates per group. Different drugs were added to the cells according to the different groups, and the cells were incubated for 48 h. After culture, the supernatant was collected, and floating cells were collected by centrifugation. Cells were digested with trypsin and combined, then washed three times with PBS buffer before use. 1× Binding Buffer was prepared, and Annexin V-Cy5 and PI double staining working solution were prepared using 1× Binding Buffer. Finally, the cells were resuspended in 300 μL PBS and incubated at 4°C in the dark for 30 min before flow cytometry was used to detect cell apoptosis.

[0089] The anti-apoptotic effect when used in combination. Statistical analysis was performed using SPSS 25.0, and the results are as follows: Figure 4 As shown, compared with the free drug combination group, the necrosis rate of the BSA-UC / Cur drug-treated group was significantly reduced (P<0.01), which may be due to the reduction of the toxicity of the free drug to cells after being made into nanoparticles. Compared with each drug-treated group, the BSA-UC / Cur drug showed the most significant reduction in mortality (P<0.01), suggesting that BSA-UC / Cur has a significant protective effect against NRK-52e-induced damage caused by inflammatory culture medium.

[0090] Experimental Example 5: Effect of nanoparticles on ROS levels of NRK-52e treated with inflammatory culture medium.

[0091] NRK-52e cells in good growth condition were collected, digested, counted, and diluted to a density of 1.5 × 10⁻⁶ cells. 5 Cell suspension at 2 ml / well was seeded into 3-well plates. Cells were incubated at 37°C with 5% CO2 for 24 h, and observed under a microscope to ensure cell adhesion and coverage of the well bottom. The old culture medium was discarded, and 2 ml of inflammatory medium was added for 1 h of stimulation. Cells were divided into PBS group, Model group, Cas-Cur group, UC group, UC+Cas-Cur group, and BSA-UC / Cur group, with 3 replicates per group. Different drugs were added to the cells according to the different groups, and the cells were incubated for 48 h. After culture, the cells were washed three times with PBS buffer, digested with trypsin, centrifuged, and then incubated at 37°C in the dark for 30 min with the ROS probe CellROX added. After incubation, the cells were washed three times with PBS buffer, resuspended in 300 μL PBS, and ROS levels were analyzed by flow cytometry.

[0092] Once transported into cells, UA acts as a pro-oxidant, contrary to its extracellular antioxidant effects, increasing the production of reactive oxygen species (ROS). Intracellular UA exerts its pro-oxidative effect solely by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Flow cytometry was used to detect the effect of nanoparticles on intracellular ROS levels in NRK-52e cells. The oxidative stress deep red fluorescent probe (CellROX) used in the experiment was non-fluorescent in its reduced state, but produced strong red fluorescence upon contact with highly oxidizing substances such as intracellular reactive oxygen species. Statistical analysis showed that... Figure 5 The median fluorescent intensity (MFI) in the Model group was significantly higher than that in the PBS group (P<0.01). This is because UA transported to cells causes mitochondrial damage, leading to increased cellular ROS levels. Compared with other drug-treated groups, the BSA-UC / Cur drug-treated group significantly downregulated ROS levels (P<0.01), thereby achieving a cell-protective effect.

[0093] Experimental Example 6: Effect of nanoparticles on mitochondrial activity in NRK-52e cells treated with inflammatory culture medium.

[0094] After establishing the inflammation model as described in Experiment 5, cells were divided into four groups: PBS group, Model group, Cas-Cur group, UC group, UC+Cas-Cur group, and BSA-UC / Cur group, with three replicates for each group. Different drugs were added to the cells according to the different groups, and the cells were incubated for 48 hours. After culture, the cells were washed three times with PBS buffer, digested with trypsin, centrifuged, and then incubated at 37°C in the dark for 30 minutes with the addition of the live-cell mitochondrial fluorescent probe, Mito Tracker. After incubation, the cells were washed three times with PBS, resuspended in 300 μL PBS, and mitochondrial activity was detected by flow cytometry.

[0095] When amino acids (UA) are taken up by cells, mitochondria are stimulated by UA, which may alter the mitochondrial membrane potential, leading to decreased mitochondrial activity and increased ROS levels. Mitochondria not only provide ATP but also play a crucial role in ROS production, making the measurement of mitochondrial activity essential. Statistical analysis showed the following results: Figure 6 As shown, the MFI in the model group was significantly enhanced compared to the control group, with a statistically significant difference (P < 0.01). The UC treatment group and the UC+Cas-Cur group showed improvement compared to the model group, but were still inferior to the BSA-UC / Cur group. The MFI in the BSA-UC / Cur treatment group was significantly higher than that in the other treatment groups (P < 0.01), indicating that BSA-UC / Cur can significantly improve mitochondrial damage induced by inflammatory culture medium.

[0096] Experimental Example 7: Effects of nanoparticles on the secretion of TNF-α, IL-6, and IL-1β by NRK-52e in inflammatory culture medium.

[0097] After establishing the inflammation model as described in Experiment 5, cells were divided into four groups: PBS group, Model group, Cas-Cur group, UC group, UC+Cas-Cur group, and BSA-UC / Cur group, with three replicates for each group. Different drugs were added to the cells according to the different groups, and the cells were incubated for 48 hours. After culture, the cell supernatant was collected and stored at -80°C for ELISA experiments. The samples were diluted 10-fold, and the levels of TNF-α, IL-6, and IL-1β were measured.

[0098] The levels of TNF-α, IL-6, and IL-β in the supernatant of NRK-52e cells were detected. The levels of TNF-α, IL-6, and IL-β were calculated based on a standard curve. Statistical analysis showed that the p-values ​​for each group of TNF-α, IL-6, and IL-β were >0.05, indicating a normal distribution. However, the p-values ​​for homogeneity of variance were <0.05, indicating that homogeneity of variance was not met. Therefore, the Dunnett-t3 test was used for pairwise comparisons between groups. The results are as follows: Figure 7 As shown, compared with the control group, the model group showed a significant increase in TNF-α, IL-6, and IL-β (P < 0.01), indicating a statistically significant difference. Compared with the other treatment groups, BSA-UC / Cur showed a significant reduction in TNF-α, IL-6, and IL-β (P < 0.01), indicating a statistically significant difference.

[0099] Experimental Example 8: Therapeutic effect of nanoparticles on a rat model of hyperuricemic kidney injury.

[0100] SPF-grade SD rats (weighing 190-200g) were used to establish a uric acid model. The rats were divided into 7 groups. Except for the Normal group, the other groups were administered potassium oxonate 400mg / kg intraperitoneally and hypoxanthine 1g / kg by gavage. Four hours after model establishment, the rats were randomly divided into 7 groups of 6 rats each: Normal group, Model group, Cas-Cur group, UC group, UC+Cas-Cur group, UC / Cur group, and BSA-UC / Cur group. The corresponding drugs were administered via tail vein according to the group, maintaining the dosage of Cur and UC at 0.5mg / kg and 1.25mg / kg, respectively. Blood samples were collected at 0, 4, 4.5, 5, 6, 7, and 8 hours before model establishment. After centrifugation, the supernatant serum was separated, and the concentration of uric acid (UA) in the serum was measured using a biochemical analyzer. A uric acid-serum curve was plotted.

[0101] SPF-grade SD rats (weighing 190-200g) were used to establish a hyperuricemic nephropathy model. The rats were divided into 7 groups. Except for the Normal group, the other groups were administered potassium oxonate 400mg / kg intraperitoneally and hypoxanthine 1g / kg by gavage to induce a model of hyperuricemic nephropathy in rats after one week. The rats were then randomly divided into 7 groups of 6 rats each: Normal group, Model group, Cas-Cur group, UC group, UC+Cas-Cur group, UC / Cur group, and BSA-UC / Cur group. From day 7, except for the Normal group, the model was continued. Four hours after model induction, the appropriate drugs were administered to each group, maintaining Cur and UC concentrations at 0.5mg / kg and 1.25mg / kg, respectively. The Normal group received the same dose of PBS for 7 days. At day 14, rats exhibiting organ dysfunction, inability to eat or drink, or weight loss reaching 25% of their original body weight were euthanized with carbon dioxide, and their organs were dissected and collected. At the end of the experiment, the animals were euthanized with carbon dioxide, and their kidneys were collected and preserved in 4% paraformaldehyde for later use. HE sections were prepared from the kidneys and subjected to pathological analysis.

[0102] An important indicator in the SD rat hyperuricemia model is the serum uric acid concentration (SUA). Hypoxanthine can increase purine concentration, thereby increasing serum UA in rats. Potassium oxonate can inhibit uricase in rats, thus inhibiting the breakdown of uric acid in the rat blood. Results are as follows: Figure 8 As shown, the SUA levels in the normal group were all less than 100 μmol / L. After modeling, serum uric acid levels in all groups increased significantly, and the SUA levels in the model group were all greater than 500 μmol / L 8 hours after modeling, proving that the modeling was successful. 0.5 hours after administration, SUA levels in the URI-containing groups decreased to below normal levels (UC, UC+Cas-Cur, UC / Cur, and BSA-UC / Cur). When 1 hour had elapsed, except for the nanoparticle group (UC / Cur and BSA-UC / Cur), the efficacy of the URI-containing groups began to decrease and return to normal SUA levels. Statistical analysis of serum UA levels 4 hours after administration showed that the normality test result was P > 0.05, indicating a normal distribution. However, the homogeneity of variance test showed P < 0.05 for each group, indicating that homogeneity of variance was not met. The Dunnett-t3 test was used for pairwise comparisons between groups. The results are as follows... Figure 8 As shown, the BSA-UC / Cur group significantly reduced SUA levels compared to the Cas-Cur, UC, and UC+Cas-Cur groups (P<0.01). Due to large intragroup differences in the UC / Cur group, there was no statistically significant difference compared to the BSA-UC / Cur group (P>0.05). However, from... Figure 8 The trend suggests that BSA-UC / Cur reduces UA for a significantly longer time than UC / Cur.

[0103] The pathological condition of kidney sections was observed by hematoxylin-eosin (HE) staining. The sections were examined under 200× and 400× microscopes. The results are as follows: Figures 9-16 As shown, in the Normal group, the proximal convoluted tubules were more numerous in cross-section, with thicker walls and smaller, irregular lumens. The epithelial cells of the proximal tubules were a single layer of cuboidal or pyramidal cells, which were relatively large and had strongly eosinophilic cytoplasm, giving them a deep red color. The brush border on the free surface was rough and uneven. The distal tubules were fewer in cross-section, with larger and more regular lumens, also surrounded by a single layer of cuboidal epithelium. The cells were smaller, neatly arranged, and had weaker eosinophilic cytoplasm, giving them a lighter red color. The free surface lacked a brush border. Considering a nephron as a single observation object, compared to the Normal group, the Model group showed significant protein accumulation in Bowman's capsule, causing obstruction. The proximal convoluted tubules exhibited more vacuolation, and mitochondria were swollen and showed protein deposition, suggesting kidney damage and successful establishment of a hyperuricemic nephropathy model. After treatment with the Cas-Cur and UC groups, Bowman's capsule protein decreased, and the vacuolation of the proximal tubules improved, but the degree of mitochondrial swelling did not significantly improve. After treatment with the Cas-Cur and UC / Cur groups, there were relatively fewer vacuoles in the proximal tubules, and protein levels in Bowman's capsule and proximal convoluted tubule were lower compared to the UC+Cas-Cur and UC groups, but mitochondrial swelling was not improved. After intervention in the BSA-UC / Cur group, there was almost no protein deposition in Bowman's capsule and proximal tubules, no vacuoles were observed, and mitochondrial swelling was significantly improved. In conclusion, compared with the other treatment groups, BSA-UC / Cur nanoparticle intervention has a significant protective effect against kidney damage.

[0104] Figures 10-16 In the diagram, small arrows represent vacuoles; large arrows represent proteins; and double arrows represent mitochondrial swelling.

[0105] It should be noted that the statistical methods used in the embodiments and experimental examples of this invention are as follows: IBM SPSS Statistics 25.0 statistical software was used for experimental data analysis. The Shapiro-Wilk test was used for quantitative data. Data conforming to a normal distribution (P>0.05) were statistically described using the mean and standard deviation (±s) and plotted as bar charts or scatter plots. Data not conforming to a normal distribution (P<0.05) were statistically described using the M (P25~P75) percentiles and plotted as box plots. Two groups of independent quantitative data conforming to a normal distribution and homogeneity of variance were tested using an independent samples t-test; two groups of quantitative data not conforming to a normal distribution and homogeneity of variance were tested using a nonparametric Mann-Whitney U test. For multiple groups of measurement data that conform to a normal distribution, one-way ANOVA is used. First, a homogeneity of variance test is performed. If the variances are homogeneous (P>0.05), the Tukey test (for more than four groups) is used for pairwise comparisons between groups. If the variances are not homogeneous (P<0.05), the Welch test is used, followed by the Dunnett-t3 test for pairwise comparisons between groups. For multiple groups of measurement data that do not conform to a normal distribution, the nonparametric rank-sum Kruskal-Wallis test is used.

[0106] The statistical results above were considered statistically significant with P < 0.05 and highly significant with P < 0.01.

[0107] In summary, the multi-enzyme-encapsulated nanoparticles of the present invention can improve the problem of cell damage caused by excessive accumulation of H2O2.

[0108] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanoparticle encapsulating multiple enzymes, characterized in that, The nanoparticles carrying multiple enzymes include core particles and a shell covering the core particles; wherein, the core particles include small molecule drug particles, uricase, and catalase, and the shell is a cross-linking layer; the small molecule drug particles are curcumin protein particles; the cross-linking layer is a tannic acid / iron polymer layer; the mass ratio of enzyme to small molecule drug particles is 50:10-50:20; The method for preparing the nanoparticles loaded with multiple enzymes includes: Drug nanoparticles and a mixed enzyme solution were mixed with a saturated salt solution to obtain metastable particles; wherein, The method for preparing the drug nanoparticles includes: mixing a drug solution with a carrier and sonicating the mixture, wherein the carrier includes casein. The metastable particles are cured with a crosslinking agent, wherein the crosslinking agent is tannic acid and ferric chloride; Incubation is performed using an incubator in which the target protein is thiolated albumin.

2. The nanoparticles for multi-enzyme coupling according to claim 1, characterized in that, The nanoparticles carrying the multi-enzyme combination have a particle size of 230-250 nm.

3. The method for preparing nanoparticles loaded with multiple enzymes as described in any one of claims 1-2, characterized in that, include: Drug nanoparticles and a mixed enzyme solution were mixed with a saturated salt solution to obtain metastable particles; wherein, The method for preparing the drug nanoparticles includes: mixing a drug solution with a carrier and sonicating the mixture, wherein the carrier includes casein. The metastable particles are cured with a crosslinking agent, wherein the crosslinking agent is tannic acid and ferric chloride; Incubation is performed using an incubator in which the target protein is thiolated albumin.

4. The method for preparing nanoparticles loaded with multiple enzymes according to claim 3, characterized in that, The saturated salt solution includes at least one of ammonium sulfate solution, potassium sulfate solution, potassium phosphate solution, and sodium chloride solution.

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